A knowledge of the mechanisms of transmitter action is essential to achieving a detailed understanding of the pathophysiology of nervous system disease, and formulating means of treatment. While much is known of the function of classical neurotransmitters, the actions of neuropeptides are less clear. I propose to study the electrophysiological response of opioid receptor activation to determine the cellular and molecular bases of opioid function. The role of opioid transmitters in endogenous mechanisms of analgesia, and in aspects of behavior, make this research clinically important. In many systems opioid peptides inhibit transmitter release. In dorsal root ganglion cells and myenteric plexus neurons opioid receptors mediate presynaptic inhibition; in these systems delta and mu-opioid receptors seem to increase a potassium current and differ from kappa-opioid receptors which apparently diminish a calcium current. No experiments have been done using voltage-clamp techniques to verify these results. I will study the electrophysiological response of the neuroblastoma x glioma hybrid cell line NO108-15 cells to opioid stimulation. These cells possess a homogeneous population of delta-opioid receptors, are well suited to detailed electrophysiological investigation, and are a model system for the study of opioid agonist pharmacology and biochemistry. Using gigohm-seal techniques, I will voltage clamp the cells to determine the ionic currents affected by delta-opioid stimulation. By use of patch-clamp recording I will measure the changes in single-channel function mediated by opioids, thus determining the fundamental electrophysiological effects of these neurotransmitters. Opioid peptides are classified as modulatory transmitters because of their known inhibition of adenylate cyclase activity and cyclic adenosine monophosphate concentration. The electrophysiological actions of opioid stimulation, however, are not clearly related to the change in cyclic nucleotide levels. By studying the relationship of these two responses of opioid stimulation in NO108-15 cells I will elucidate the mechanisms responsible for the electrophysiological changes. Chronic opioid and chronic ethanol exposure of NO108-15 cells both produce changes in delta-opioid receptor function. The cells appear to demonstrate tolerance and dependence to these two substances. No cellular physiological investigation has yet characterized the features of cells either tolerant to opioid or ethanol application, or in the state of acute withdrawal. The electrophysiological changes associated with these states, which have been studied pharmacologically and biochemically, will be determined. These studies will provide further insights into the mechanisms of action of the opioid receptor.